Interactions of Heteroaromatic Compounds with Nucleic Acids

Abstract

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, "Hoechst 33258" and two acridines substituted with t-butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations.Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA.The main results from DNA binding studies indicate that the triphenylmethane dyes except p-fuchsin are bound with high preference to two adjacent A T pairs while Hoechst 33258 seems to need three A -T pairs as the binding site.Model studies with synthetic polynucleotides revealed that not only a sequence of A . T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA.Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.We were able to show recently that tricyclic ring systems of high polarisability which interact with DNA by intercalation show either no base specificity or G . C specificity [1,2]. The fact that we never observed A . T specificity for DNA ligands of this type led us to assume that a specific interaction with A . T base pairs might be basically different in type from the intercalation reaction. This view was supported by the fact that all known A . T-specific DNA ligands other than metal ions are apparently bound to the outside of the DNA helix. This holds for the tetraalkylammonium salts [3], for netropsin and distamycin antibiotics [4], as well as for hydroxystilbamidine [5]. Our finding that the weak G C specificity of proflavine [l] is transformed to a strong A T specificity if the ring system is prevented from intercalation by steric hindrance [6] points in the same direction.In order to obtain further experimental evidence for the above hypothesis, we studied the base specificity of other DNA ligands, such as dyes used for staining nuclei or chromosomes, for which the formation of intercalation complexes with DNA seemed improbable due to their chemical structures. To this class of compounds belong the common triphenylmethane dyes such as p-fuchsin, methyl green, crystal violet and malachite green, as well as auramine and the bisbenzimidazol derivative "Hoechst 33258"; the latter was introduced for chromosome staining techniques by Herzog and Schiitze The main result of the present study is in full agreement with our expectations. None of the compounds shows G . C specificity; they all show pronounced A . T specificities, except p-fuchsin which is unspecific. In this property these dyes behave similarly to di-tbutyl-proflavine [6]. This led us to include two derivatives of the latter in th...

Interactions between pairs of DNA‐binding dyes: Results and implications for chromosome analysis

“…As previously reported (22, 36, 37), a reverse (R-band) staining pattern is produced if a G-C specific energy donor (chromomycin A3) (3) is used together with an A-T specific M 33258 Hoechst ( A ), 33258 Hoechst followed by energy acceptor (methyl green) (33). Chromosomal regions exhibiting dull quinacrine fluorescence (e.g., most of human chromosome 22) fluoresce brightly with this latter combination.…”

Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D

“…H33258 did not compete for 7-AMD binding sites, since similar results (546-nm excitation) were obtained when H33258 was omitted from the staining buffer. This was to be expected, since 7-AMD shows G-C specificity (16,19,25), while H33258 preferentially binds to A-T stretches (16,20,25). It has been shown that H33258 and 7-AMD do not compete for binding sites when bound to DNA (16,25,. Seventy percent ethanol-fixed cells were stained with acridine orange (Fluka) in 0.15 M NaC1, 5 mM EDTA, 10 mM Tris-HCl (pH = 7.4) at < 0.6 -lo6 cells/ml after one wash in the same buffer.…”

“…1 The results of the DNasel experiments are also intriguing and indicate that 7-AMD binding in formaldehydefixed chromatin of leucocytes is restricted by the topological state of DNA (i.e., that the double-stranded DNA is quasicircular). Binding of 7-AMD, a n intercalator, increased after mild DNasel digestion, while the binding of H33258, a probe which binds externally along three A-T basepairs (20), decreased monotonically. Binding of intercalators at high concentrations is assumed to increase after nicking of DNA (3,24), which may explain the enhanced levels of bound 7-AMD in DNasel-digested chromatin.…”

Interctions of pyronin Y(G) with nucleic acids

“…However, in view of the Miiller and Crothers' study (21) indicating G.C specificity, the most likely explanation for low $JR of natural DNA is preferential intercalation of PY between the base pairs containing guanine at this low binding density, rather than the long-distance effects of this base.…”